A K‐RAS Inhibitor Abrogates Self‐Renewal of Pancreatic Cancer Stem Cells via K‐RAS – NF‐κB – SOX2 Axis

Abstract

BackgroundGrowing evidence indicate that pancreatic cancer stem cells (pCSCs) being the culprit of cancer recurrence and metastasis. These chemoresistant cells remain undifferentiated, while giving rise to their differentiated progenies in an asymmetrical cell division known as self‐renewal to sustain tumor maintenance and progression. Therefore, targeting the self‐renewal of pCSCs is no doubt a key strategy to eradicate them. Here, we are the first to describe 4,6‐dichloro‐2‐methyl‐3‐aminoethyl‐indole (DCAI), a known K‐RAS binder that inhibits K‐RAS signaling, as a novel anti‐pCSC agent; its mechanism of action in abrogating the self‐renewal of pCSCs was elucidated.MethodsCell viability assay was used to determine the chemoresistantance of pancreatic cancer cell lines with clinically relevant K‐RAS mutations: PANC‐1 (K‐RASG12D), Capan‐2 (K‐RASG12V), and MIA PaCa‐2 (K‐RASG12C). Tumorsphere assay, which is a hallmark test platform for evaluating potential self‐renewal inhibitors in vitro was utilized. We employed this assay to investigate the effect of DCAI on the self‐renewal of pCSCs. Briefly, tumorspheres were treated with test compounds, followed by counting intact tumorspheres (diameter ≥ 60 μm). The molecular players of stem cell maintenance, K‐RAS and NF‐κB pathways in tumorspheres, before and after treatments, were examined by Western blotting.ResultsCapan‐2 (GI25 > 100 μM) and PANC‐1 (GI25 = 90 ± 17 μM) were equally ranked as the most resistant cell line to gemcitabine, as compared with MIA PaCa‐2 (GI25 = 0.053 ± 0.009 μM) cells. This profound chemoresistance coincided with the tumorsphere‐forming ability, which was found solely in Capan‐2. The Capan‐2 tumourspheres were remarkably irresponsive to gemcitabine (IC50 > 100 μM). We were intrigued to learn that DCAI (IC50 = 5 μM) abrogated the formation of Capan‐2 tumorspheres more effectively than vismodegib (IC50 > 20 μM), an anti‐pCSC agent. Several studies collectively suggested a link between K‐RAS signaling, NF‐κB signaling, and SOX2 expression. In our study, Capan‐2 tumourspheres demonstrated hyperactivations of MAPK and AKT pathways, both of which are downstream of K‐RAS. Activation of P65 NF‐κB signaling molecule was observed in respond to the hyperactivation of K‐RAS signaling, which eventually led to the overexpression of SOX2. Upon treatment with DCAI, K‐RAS and NF‐κB signalings were significantly inhibited and SOX2 expression was greatly reduced in Capan‐2 tumourspheres.ConclusionOur present study revealed that DCAI is a promising agent pertaining to targeting the self‐renewal of pCSCs via K‐RAS – NF‐κB – SOX2 axis. It is suggested that K‐RAS is a valid therapeutic target in pCSCs for eradicating pancreatic cancer recurrence and metastasis. Targeting K‐RAS could form a solution to the problem that remains unsolved for many decades.Support or Funding InformationMalaysian Ministry of Education (Fundamental Research Grant Scheme, 04‐01‐17‐1905FR)